The invention provides nucleotide sequences coding for the accDA gene and a process for the preparation of L-amino acids, especially L-lysine, by fermentation using corynebacteria in which the accDA gene is amplified.
L-Amino acids, especially L-lysine, are used in animal nutrition, in human medicine and in the pharmaceutical industry.
It is known that these amino acids are prepared by the fermentation of strains of corynebacteria, especially Corynebacterium glutamicum. Because of their great importance, attempts are constantly being made to improve the preparative processes. Improvements to the processes may relate to measures involving the fermentation technology, e.g. stirring and oxygen supply, or the composition of the nutrient media, e.g. the sugar concentration during fermentation, or the work-up to the product form, e.g. by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself.
The productivity characteristics of these microorganisms are improved by using methods of mutagenesis, selection and mutant choice to give strains which are resistant to antimetabolites, e.g. the lysine analog S-(2-aminoethyl) cysteine, or auxotrophic for amino acids of regulatory significance, and produce L-amino acids.
Methods of recombinant DNA technology have also been used for some years in order to improve L-amino acid-producing strains of Corynebacterium by amplifying individual amino acid biosynthesis genes and studying the effect on L-lysine production. Surveys of this subject have been published inter alia by Kinoshita (xe2x80x9cGlutamic Acid Bacteriaxe2x80x9d in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6, 261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
The enzyme acetyl-CoA carboxylase catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. The enzyme from Escherichia coli consists of four subunits. The accB gene codes for biotin carboxyl carrier protein, the accC gene for biotin carboxylase and the accA and accD genes for transcarboxylase (Cronan and Rock, Biosynthesis of Membrane Lipids, in: Escherichia coli and Salmonella typhimurium (ed. F. C. Neidhardt), 1996, pp. 612-636, American Society for Microbiology). Because of the property of the enzyme to carboxylate acyl groups in the form of acyl-CoA, it is also called acyl-CoA carboxylase.
The nucleotide sequence of the accBC gene from Corynebacterium glutamicum has been determined by J{overscore (a)}ger et al. (Archives of Microbiology 166, 76-82 (1996)) and is generally available from the data bank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) under accession number U35023. The accBC gene codes for a subunit of acetyl-CoA carboxylase which carries a biotin carboxyl carrier protein domain and a biotin carboxylase domain.
The object which the inventors set themselves was to provide novel procedures for the improved preparation of L-amino acids, especially L-lysine, by fermentation.